1. Field of the Invention
The invention relates generally to the fields of medicine and cell biology and, more specifically, to the fields of infectious disease and regulation of apoptosis and inflammation.
2. Background Information
Post-translational modification of proteins is an important means of regulating protein activity, stability or localization. For example, post-translational modification of target proteins by conjugation to the small protein ubiquitin earmarks the target protein for degradation by the 26S proteasome. Recently, several small proteins have been identified with sequence similarity to ubiquitin and which modify target proteins. These ubiquitin-like modifiers (UBLs) include SUMO (small ubiquitin-related modifier), Rub1 (also called Nedd8), Apg8 and Apg12. In mammals, three members of the SUMO family have been described: SUMO-1, also known as PIC-1, sentrin or GMP1, which in humans is a 101 amino acid polypeptide; and the highly homologous polypeptides SUMO-2 and SUMO-3. Although SUMO-1 shares only about 18% sequence identity to ubiquitin, both polypeptides share a common three-dimensional structure.
The pathway of protein modification by sumoylation is analogous to the well-characterized pathway of modification by ubiquitination, although a different set of enzymes are involved. SUMO is initially made as an inactive precursor. The precursor is then processed by proteolytic cleavage to yield the active modifier polypeptide with an exposed carboxy-terminal glycine residue. The exposed glycine is required for the formation of an isopeptide bond between the carboxyl terminus of SUMO and a lysine residue of the target protein. This SUMO processing reaction is catalyzed by a cysteine protease known as a SUMO-specific protease. An E1-type activating enzyme, an E2-type conjugating enzyme, and an as-yet-unidentified E3-type ligase enzyme, are sequentially required for the conjugation of the processed SUMO to the target protein. The SUMO-specific protease that processes SUMO can also catalyze the cleavage of conjugated SUMO from the target protein.
Several known SUMO substrates are important modulators of apoptosis. Apoptosis, or programmed cell death, is involved in the development and homeostasis of multicellular organisms. Additionally, apoptosis of infected cells provides the host organism with an effective defense mechanism against pathogens. Alterations in the normal process of apoptosis occur in various pathological conditions, including cancer, autoimmune diseases, inflammatory conditions, degenerative syndromes and infectious diseases.
One SUMO target that plays a key role in apoptosis is the Promyelocytic Leukemia protein, or PML. The assembly and/or stability of PML nuclear bodies (PML NBs or PODs) is modulated by sumoylation of PML. It has been proposed that PML NBs are potential sites of protein degradation. Although the functions of PML NBs have not been fully defined, it is recognized that assembly of PML NBs is sensitive to environmental stimuli, and is compromised in pathological situations such as certain cancers and infectious disorders. PML NBs contain a number of proteins that are transiently recruited to the nuclear body, including the pro-apoptotic protein p53, various transcriptional regulators and proteasome components.
Another SUMO target that plays a key role in apoptosis is the inhibitor of the transcription factor NFκB, known as IκBα. NFκB is kept in an inactive form in the cytosol by binding to IκBα. Stimulation of the cell with various effectors, such as pro-inflammatory cytokines, various infectious agents and environmental stresses, leads to IκBα phosphorylation, ubiquitination and ultimately proteolytic degradation. NFκB is thus liberated from its inhibitor to enter the nucleus and activate its target genes, which include anti-apoptotic genes and genes involved in immune and inflammatory responses. SUMO competes with ubiquitin for modification of IκBα, as both modifiers target the same lysine residue of IκBα. The SUMO-modified pool of IκBα is protected from degradation, and the sumoylation of IκBα thus inhibits NFκB function.
A protein from the bacterial pathogen Yersinia pestis, YopJ, which is essential for virulence, has been shown to be a SUMO-specific protease (Orth et al., Science 290:1594-1597 (2000)). YopJ exerts its pathogenic effects on cells by disrupting post-translational modifications of a number of cellular substrates involved in the production of immune cytokines and anti-apoptotic factors. For example, YopJ expression prevents activation of the MAPK pathway and the NFκB pathway in the host, whereas catalytic domain mutants of YopJ do not affect these pathways. Therefore, SUMO-like protease activity is critical for microbial pathogenicity and host immune responses.
In view of the important role of SUMO-specific proteases in apoptosis, inflammation, host defenses against infectious agents, and other biological processes, there exists a need to identify novel microbial SUMO-specific proteases and molecules that regulate sumoylation. Such proteases and regulatory molecules can be used in the development of antibiotics, as well as in the development of therapeutic agents for the treatment of disorders of apoptotic regulation. The present invention satisfies this need and provides related advantages as well.